A tissue culture method recently has been established in which cultures consisting almost entirely of human melanocytes can be obtained from epidermal single cell suspensions by the use of 12-0-tetradecanoyl phorbol 13-acetate (TPA) in the culture medium. Growth of melanocytes was achieved in a medium containing cholera toxin and TPA. Pure cultures of melanocytes were obtained by immuno-rosetting with monoclonal antibodies combined with Percoll gradients. It is proposed to use this in vitro system: (1)\to characterize the newly established melanocyte populations and to confirm their normal status; (2)\to investigate the regulation of growth and differentiation of human melanocytes by different hormones and factors, i.e., melanocyte stimulation hormone (MSH), epidermal growth factor (EGF), nerve growth factor (NGF), fibroblast growth factor (FGF), TPA and other tumor promotor (i.e., mezerein and teleocidin), cholera toxin, cAMP and cGMP; and (3)\To develop monoclonal antibodies to human melanocyte cell surface antigens. The effect of different factors in melanocyte growth and differentiation will be evaluated by light microscopy combined with histochemical staining procedures, biochemical assays for tyrosinase, flow cytometry combined with autoradiography and transmission electron-microscopy and by cell surface markers. Detailed chromosomal analysis will be carried out on cells grown under various culture conditions, with special emphasis on melanocytes grown in the presence of tumor promotors. The monoclonal antibodies will be used to serologically characterize melanocytedifferentiation antigens, to isolate melanocyte and melanocyte precursor cell populations, and for immuno-electromicroscopy.